Red tide assemblage formation in an estuarine upwelling ecosystem: Ria de Vigo

Red tides are conspicuous in the upwelling system of Galicia(NW Iberian Peninsula). At present, there are conflicting hypothesesabout the generation site of these phytoplankton assemblages.It is interesting to know whether the rias can be sites of redtide formation or if they act only as accumulation sites ofpopulations advected from shelf waters. A study in the Ra deVigo, carried out during late September 1990, showed the developmentof a red tide assemblage, composed of Alexandrium affinis, Ceraiiumfusus and Gymnodinium catenaium, during a 2 week upwelling-downwellingcycle. Growth occurred at the bottom of the thermocline-topof the nutricline. Above this assemblage, a diatom assemblage(large diatoms) was blooming. Prior to the formation of thered tide, a subsurface chlorophyll maximum made up of smalldiatoms (Nilzschia f. seriaia, Chaeloceros socialis), smallflagellates (<30 µm) and small gymnodinid forms (<30µm) was observed. In the nutrient-depleted upper layer,several autotrophic and large heterotrophic dinoflagellatesdominated. It is suggested that the ratio between the velocityof upward water movement and the depth of the stratified upperlayer (flushing rate, day^–1) is the critical parameterwhich triggers active phytoplankton growth. It can be concludedthat upward water velocities of {small tilde}2.5 m day^–1and a stratified upper layer of 10 m depth (flushing rate 0.25day^–1) are the main physical constraints for red tidedevelopment.     

Diurnal vertical migration of Gymnodinium mikimotoi during a red tide in Hoketsu Bay, Japan

The depth, velocity and timing of the vertical migration ofGymnodinium mikimotoi were examined using fluorometric fieldobservations. Gymnodinium mikimotoi can migrate vertically {smalltilde}20 m daily at velocities of {small tilde}2.2 m h^–1     

Changes in bacterial populations during red tides caused by Mesodinium rubrum and Gymnodinium catenatum in North West Coast of Spain

Heterotrophic bacterial communities associated with four red tides caused by Mesodinium rubrum and Gymnodinium catenatum in two Galician Rias (North West Spain) were examined. Three of these were produced by the Mesodinium rubrum and the causative organism of a toxic bloom was Gymnodinium catenatum. In early stages of all the blooms, the diversity decreased but the total marine bacterial counts increased by one or two logs. Vibrio numbers were also incremented by two logs in two blooms of M. rubrum, while in the other bloom of this organism and in the red tide caused by G. catenatum a decrease in number of these bacteria was observed. A total of 116 bacterial strains were identified at the genus level and grouped into 12 phena. During the decomposition processes of two blooms of M. rubrum a zooplanktonic-type bacterial succession was observed (Vibrio, pseudomonads and Moraxella-Acinetobacter). On the other hand, during decomposition of the other red tide of M. rubrum and the bloom of G. catenatum, a typical phytoplanktonic-type succession occurred, as Pseudomonas and Moraxella groups became dominant for all the process. These results support the conflicting taxonomical position of M. rubrum. After the blooms, the changes in the community point towards the restablishment of the normal bacterial flora of the estuary (increase in diversity and decreases of bacterial numbers). Only the Vibrio strains, isolated from the non-toxic first and second red tides, displayed cytotoxic activities. A relationship among bacterial cytotoxicity and toxic effects of blooms cannot therefore be established.     

Changes in bacterial populations during red tides caused by Mesodinium rubrum and Gymnodinium catenatum in North West Coast of Spain

Heterotrophic bacterial communities associated with four red tides caused by Mesodinium rubrum and Gymnodinium catenatum in two Galician Rias (North West Spain) were examined. Three of these were produced by the Mesodinium rubrum and the causative organism of a toxic bloom was Gymnodinium catenatum . In early stages of all the blooms, the diversity decreased but the total marine bacterial counts increased by one or two logs. Vibrio numbers were also incremented by two logs in two blooms of M. rubrum , while in the other bloom of this organism and in the red tide caused by G. catenatum a decrease in number of these bacteria was observed. A total of 116 bacterial strains were identified at the genus level and grouped into 12 phena. During the decomposition processes of two blooms of M. rubrum a zooplanktonic-type bacterial succession was observed ( Vibrio , pseudomonads and Moraxella-Acinetobacter ). On the other hand, during decomposition of the other red tide of M. rubrum and the bloom of G. catenatum , a typical phytoplanktonic-type succession occurred, as Pseudomonas and Moraxella groups became dominant for all the process. These results support the conflicting taxonomical position of M. rubrum . After the blooms, the changes in the community point towards the restablishment of the normal bacterial flora of the estuary (increase in diversity and decreases of bacterial numbers). Only the Vibrio strains, isolated from the non-toxic first and second red tides, displayed cytotoxic activities. A relationship among bacterial cytotoxicity and toxic effects of blooms cannot therefore be established.     

Presence of Gymnodinium catenatum (Dinophyceae) in a coastal Mediterranean lagoon

The occurrence and abundance of the toxic, chain-forming dinoflagellateGymnodinium catenatum in a Tyrrhenian coastal lagoon, the Fusaro,during an annual sampling cycle are reported. Peak abundanceswere observed from late spring until early autumn Although veryhigh cell numbers were recorded, up to 1 5 x 10⁶ cells l^–1,no monospecific bloom of this species occurred. The first observationof G.catenatum in the Mediterranean occurred in the Fusaro andthe appearance of this species in a traditional shellfish farmingarea, where no shellfish intoxication has been reported to date,is discussed in relation to human interventions in the basin.In particular, intensive dredging in recent years with resuspensionof bottom sediments may have seeded the water body with cysts.A Gymnodinium n d species, illustrated using scanning electronmicroscopy, caused a monospecific bloom in concomitance withmaximum abundances of G.catenatum, apparently outcompeting thislatter species     

Possible factors responsible for the toxicity of Cochlodinium polykrikoides,a red tide phytoplankton

Cochlodinium polykrikoides, a harmful red tide dinoflagellate, is highly toxic to fish, but the toxic mechanism is still unknown. Recent study has suggested that C. polykrikoides generates reactive oxygen species (ROS) such as superoxide anion (O(2)(-)) and hydrogen peroxide (H(2)O(2)), and the ROS-mediated ichthyotoxicity has been proposed. In this study, we found that the levels of O(2)(-) and H(2)O(2) detected in C. polykrikoides were trace levels as compared with those of Chattonella marina which is well-known to produce ROS. Furthermore, no significant increase in O(2)(-) generation by C. polykrikoides was observed in the presence of lectins such as concanavalin A (Con A) and wheat germ agglutinin (WGA) or fish mucus prepared from skin and gill of yellowtail, whereas C. marina generated increased level of O(2)(-) responding to these stimuli. Interestingly, the cell-free aqueous extract prepared from C. polykrikoides showed toxic effect on the HeLa cells, but the extract of C. marina had no significant effect. Furthermore, gradual accumulation of polysaccharides in the medium was observed during the growth of C. polykrikoides, and the medium gradually became viscous, but no such changes were observed in the medium of C. marina. These results suggest that multiple factors may be responsible for the toxic mechanism of C. polykrikoides.     

Isolation and properties of isolated nuclei from the Florida red tide dinoflagellate Gymnodinium breve (Davis)

A simple and rapid method is described for the isolation of nuclei from the Florida red tide dinoflagellate Gymnodinium breve. The nuclei are free of cytoplasmic contamination and are active in endogenous RNA synthesis. The ratio of DNA : RNA : acid-soluble protein : acid-insoluble protein is 1:0.39:0.13:0.63, respectively, and each nucleus contains ca. 113 picograms of DNA. Electrophoretic analysis of the acid-soluble proteins reveals the presence of two histone-like proteins with molecular weights of 12,000 and 13,000.     

Critical micelle concentrations of allelopathic substances produced by Nannochloris oculata which affect a red tide organism, Gymnodinium breve

Laboratory cultures of the green algae Nannochloris oculata and Nannochloris eucaryotum are known to cause lysis of Gymnodinium breve, which is Florida's red tide organism. Two cytolytic agents were previously identified as methyl palmitate and methyl stearate. In this study, the critical micelle concentrations of these substances were determined by ultraviolet light and turbidimetric methods to be 3.5 +/- 0.3 ppm (methyl stearate) and 4.3 +/- 0.6 (methyl palmitate). There were no significant differences in results obtained using the two methods.     

Isolation and characterization of light-dependent hemolytic cytotoxin from harmful red tide phytoplankton Chattonella marina

Chattonella marina (C. marina), a raphidophycean flagellate, is a causative organism of red tide, and highly toxic to fish. In this study, we found that the cell-free methanol extract prepared from this flagellate exhibited potent hemolytic activity against rabbit erythrocytes. Interestingly, the hemolytic activity of the extract was absolutely light-dependent, and no hemolytic activity was detected in the dark even at very high concentration. Gel filtration chromatography of the methanol extract on a column of Sephadex LH-20 revealed that the extract contained hemagglutinin as well as hemolytic agents, and the substances responsible for these activities were separately eluted. These results suggest that the hemagglutinating and hemolytic activities were derived from distinct compounds. The hemolytic fraction obtained after gel filtration (F4) caused marked inhibition of the growth of C. marina itself and other species of phytoplanktons. Furthermore, F4 showed a potent cytotoxicity toward various mammalian cultured cell lines including human tumor cells (HeLa cells) in a dose-dependent manner. The cytotoxicity was also light-dependent, and no cytotoxic effect was exhibited in any cell lines tested in the dark. After further purification procedures via preparative thin-layer chromatography and subsequent HPLC, a major hemolytic agent was obtained as highly purified form. Since the methanol extracts prepared from other raphidophycean flagellates such as Heterosigma akashiwo, Olisthodiscus luteus, and Fibrocapsa japonica showed light-dependent hemolytic activity toward rabbit erythrocytes, it was suggested that the light-dependent hemolytic agents commonly exist at least in these raphidophycean flagellates.     

Galacturonic-acid-induced increase of superoxide production in red tide phytoplankton Chattonella marina and Heterosigma akashiwo

Red tide phytoplankton, Chattonella marina and Heterosigma akashiwo, are known to generate superoxide anion (O2-). We found that galacturonic acid (GaLUA) stimulated C. marina and H. akashiwo to generate increased amounts of O2-. Since such effect was not observed in any other monosaccharides tested, our results suggest that the binding of GalUA to specific sites on the flagellate cell surface may induce the increase of 02- production.     

Intraspecific variation in the selenium requirement of different geographic strains of the toxic dinoflagellate Gymnodinium catenatum

The requirement for selenium (IV) was assessed in five strainsof the toxic dinoflagellate Gymnodinium catenatum Graham, representingthree populations from Tasmania (Australia), as well as oneeach from Japan and Spain. Strains were grown in nutrient-enrichedsea water medium with 10^–9 M selenium added as selenite(H₂SeO₃), or with no added selenium, and monitored for growthand cell yield. Strains exhibited different selenium (Se) requirements,as evidenced by (i) a decrease in exponential growth rate (10–20%)and cell yield (up to 80%) (Japanese strain); (ii) a decreasein cell yield only (Tasmania Derwent Estuary 1987, TasmaniaHuon Estuary and Spanish strains); and (iii) no decrease ingrowth or cell yield (Tasmania Derwent Estuary 1993 strain).Variation in the response to Se deficiency was greatest betweenthe two strains isolated from the Derwent Estuary, Tasmania,in different years (1987 and 1993) and less between Tasmanianstrains from different localities (Huon and Derwent Estuariesare 50 km apart) or between Tasmanian and Japanese or Spanishstrains. Strain variability in micro-nutrient responses suchas described here may provide a partial explanation for differentbloom patterns exhibited by the same dinoflagellate taxon underapparently similar environmental conditions.     

Comparative study of selenium requirements of three phytoplankton species: Gymnodinium catenatum, Alexandrium minutum (Dinophyta) and Chaetoceros cf. tenuissimus (Bacillariophyta)

This study investigated the selenium (SE) requirements of three phytoplankton species which commonly bloom in southern Australian estuaries. The present study showed that the toxic dinoflagellate Gymnodinium catenatum Graham had an obligate requirement for Se (IV) in culture. After two transfers (4 weeks = 7 generations) in Se-deficient seawater medium, this phytoplankton species exhibited a decline in growth rate (25%) and biomass yield (90%), while complete cessation of cell division occurred under prolonged (8 weeks = 12 generations) Se starvation. Addition of 10^-9-10^-7 M H2SeO3 to nutrient-enriched seawater medium resulted in increased G.catenatum growth and biomass yields in direct proportion to the Se concentrations offered. In contrast to G.catenatum, Se limitation was observed in the dinoflagellate Alexandrium minutum Halim after four transfers (5 weeks = 20 generations) in Se-deficient medium. Exponential growth rates of A.minutum decreased slightly (5-10%) when Se was not supplied, but biomass yields decreased as much as 80-90%. The diatom Chaetoceros cf. <It>tenuissimus Meunier showed no evidence of Se limitation even after eight transfers (8 weeks; >60 generations) in Se-deficient medium. Variations in growth rates and biomass yields between transfers provide valuable information about the relative potential for Se limitation in the three species studied. In addition, differences in Se requirement between these bloom-forming phytoplankton species suggest that this micronutrient may play a role in structuring phytoplankton communities in southern Australian waters.      

Crab and mollusc shell drilling by Octopus vulgaris (Mollusca: Cephalopoda) in the Ria de Vigo (north-west Spain)

Octopus vulgaris living in the Ria de Vigo, north-west Spain, sometimes drills a cavity in the carapace of crabs and shells of molluscs. These holes have features with the same characteristics as those made in molluscs by O. vulgaris and in crabs by Eledone cirrhosa when isolated in aquaria. This is the first report of drilling the carapace of crabs by Octopus vulgaris .     

Purification and characterization of a novel high molecular weight exotoxin produced by red tide phytoplankton,Alexandrium tamarense

Our recent studies have demonstrated that the aqueous extract prepared from Alexandrium tamarense, a harmful red tide phytoplankton, showed cytotoxicity on Vero cells. In this study, the toxic substance was purified from the culture supernatant of A. tamarense. Based on the gel‐filtration profile, the molecular mass of a purified toxin was estimated to be about 1,000 kDa. On sodium dodecylsulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) analysis, a main band with molecular mass of 1,000 kDa was detected with periodic acid‐Schiff (PAS) staining, but no protein bands were detected by Coomassie brilliant blue (CBB) protein staining. Sugar composition analysis of the toxin suggested that the toxin contains galactose, fucose, mannose, N‐acetylglucosamine, xylose, and other minor saccharides, whereas no significant levels of amino acids were detected by amino acid analysis. These results suggest that the toxin is a polysaccharide‐based compound. The toxin showed cytotoxic effects on various cell lines in a concentration‐dependent manner. Among the cell lines tested, U937 cells were the most susceptible to the toxin. In U937 cells treated with the toxin, a typical apoptotic nuclear morphological change and DNA fragmentation were observed. This is the first report demonstrating that a polysaccharide‐based toxin isolated from red tide phytoplankton can induce apoptotic cell death. © 2008 Wiley Periodicals, Inc. J Biochem Mol Toxicol 22:405–415, 2008; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20253     

Food of the cuttlefish Sepia officinalis and S. elegans in the Ria de Vigo (NW Spain) (Mollusca: Cephalopoda)

A study of the diet of Sepia officinalis and S. elegans in the Ria de Vigo has shown that crustaceans are the most abundant prey in both species, followed by fish. Changes in the food composition of both species occur with growth. The type of prey eaten by the two sexes of these species is very similar. The possibility of trophic competition between juveniles of S. qficinalis and S. elegans is discussed.     

Monitoring brevetoxins during a Gymnodinium breve red tide: comparison of sodium channel specific cytotoxicity assay and mouse bioassay for determination of neurotoxic shellfish toxins in shellfish extracts

In October of 1996, a Gymnodinium breve bloom occurred in shellfish harvesting waters of Alabama, Mississippi and Louisiana, Gulf of Mexico, USA. Bloom densities reached 5.6x10(5) cells liter(-1) and bloom residence at shellfish sampling stations ranged from 3 to 28 days. Brevetoxin-2 dominated G. breve toxin profiles in bloom seawater extracts. Shellfish toxicity, assessed by mouse bioassay, exceeded the guidance level for up to 75 days after the bloom had dissipated. Cytotoxicity assays and mouse bioassays showed similar temporal patterns of shellfish toxicity, but the two methods differed in estimations of brevetoxin-3 equivalent toxicity by a factor of 93 to 1. LC-ESI-MS showed the temporal patterns in shellfish toxicity reflected metabolism of G. breve toxins. The molecular ions m/z 1004, 1017 and 1033 dominated LC-ESI-MS spectra of toxic chromatographic fractions from the extracts and were identified as brevetoxin metabolites on the basis of LC-APCI-MS-MS. The discrepancy between cytotoxicity and mouse bioassay estimates of brevetoxin-3 equivalent toxicity resulted from the difference in extraction efficiency of solvents used in the respective methods and the relative sensitivity of the assays to toxin metabolite mixtures present in the extracts. The normalized cytotoxicity assay showed 75% agreement with mouse bioassay positive test samples and 64% agreement with mouse bioassay negative test samples. Published in 1999 by John Wiley & Sons, Ltd.     

Distribution and abundance of sardine eggs in the Ria of Vigo (NW Spain), 1979-1984

The distribution and abundance of sardine eggs in the Ria ofVigo (Galician coast, NW Spain) was studied monthly for threeannual cycles. Sardine eggs were most abundant during the winterand spring months, although the spawning peaks varied from yearto year. The greatest densities of eggs were found in Novemberand March during the 1979–1980 cycle but no autumn peakwas found in 1981,1982 or 1983. The number of eggs also variesgreatly in different years. The greatest densities of eggs werealways found in the outer area of the ria. The study of thevertical distribution of sardine eggs shows that they are concentratedabove the thermocline.     

Ria de Alvor as a spawning place and a nursery ground

Ria de Alvor is a small estuary between Lagos and Portimão on the south coast of Portugal, 37°7'–37°10'N, 8°35'–8°38'W.
Samples were collected monthly in the period April 1985–March 1986 in an ichthyoplankton and ichthyofauna survey of the estuary. A WP-2 plankton net was used for catching ichthyoplankton, and a beam trawl and a beach seine were used for catching ichthyofauna.
Scaldfish, Arnoglossus laterna (Walbaum, 1792), even though the most abundant species in ichthyoplankton samples, was never found in ichthyofauna samples.
From the results obtained, it is concluded that a great part of the fish species are temporary marine visitors using the Ria as a nursery ground, although this system appears to provide a spawning area for species with benthic eggs. No evidence was found of pelagic spawning.     

Modeling the occurrence of shellfish poisoning outbreaks caused by Gymnodinium catenatum (Dinophyceae) through electromagnetic signal triggering

Accumulation of paralytic shellfish poisoning toxins (PSTs) in bivalves attributed to Gymnodinium catenatum blooms at the NW Portuguese coast was previously associated with periods of low solar activity (measured by the radio flux R), or low geomagnetic A _a index. It was also observed that reduction of R preceded the occurrence of toxin accumulation, while the A _a index increase could be related to its absence during periods of low activity. For modeling toxin accumulation, the monthly decrease in R was studied along the decade 2003–2012. A match that helped explaining the highly toxic years of 2007 and 2008 was obtained by plotting the formula: ΔR = (R _{n ? 1} ? R _n )/(R _n ? 65)², where 65 represented the lowest radio activity known to date. The complex denominator was required to take into account the sunspot cycle. A 1–2-month lag was observed between maximal relative decline and maximal PST accumulation. PSTs in bivalves from the Portuguese south coast were related with natural electromagnetic cycles for the first time, and were not statistically associated with low R. A statistically significant association with low A _a index also was not achieved, due to the low number of occurrences, although the 25–75 percentile was restricted to low A _a indexes in a similar way to that found for the NW coast. PST accumulation outside solar minima could be triggered by a steep decline in the A _a index (ΔA), but no lag was observed in this case. While the ΔR amplitude helped explaining the highly toxic years of 2007 and 2008 at the NW coast, the amplitude of ΔA was not related to the severity of the accumulation. Another kind of local electromagnetic signaling was investigated resorting to the occurrence of seismologic phenomena, because these events can trigger electric activities. No statistical association was found between seism number or magnitude and PSTs at the south coast, located near the boundary between the African and Eurasian plates, and marked by moderate seismicity.